Process for preparing cardiodilatin fragments; highly purified cardiodilatin fragments and intermediate products for the preparation of same

ABSTRACT

The invention relates to a process for the preparation of cardiodilatin fragments, to highly purified cardiodilatin fragments, and to appropriate intermediates for the preparation of said fragments. Furthermore, the invention relates to highly purified cardiodilatin fragments which are free of peptide impurities and exhibit a single migration peak in capillary electrophoresis, as well as to appropriate processes for the preparation of same.

The invention relates to a process for the preparation of cardiodilatinfragments, to highly purified cardiodilatin fragments, and toappropriate intermediates for the preparation of said fragments.

The present invention is directed to a process for the preparation ofcardiodilatin fragments of formula IR¹-ANP(105-121)-R²  (I),

having a chain length of 17-37 amino acids in total, whereinANP(105-121) represents the amino acid sequence [SEQ ID NO. 1],

-   R¹ represents an amino acid chain of sequence ANP(90-104) [SEQ ID    NO. 2] or fragments thereof having a chain length of 0-15 amino    acids, and-   R² represents an amino acid chain of sequence ANP(122-126) [SEQ ID    NO. 3] or fragments thereof having a chain length of 0-5 amino    acids,

wherein synthesis is effected via condensation of at least three partialfragments, and condensation of the partial fragments to give thecardiodilatin fragments of formula I is carried out between the aminoacid positions Gly¹⁰⁸ and Arg¹⁰⁹ and the amino acid positions Gly¹²⁰ andCys¹²¹.

Cardiodilatin is a peptide of the class of natriuretic peptides. Thesepeptides play an important role in regulating the balance of salts andwater in the body. The prototype of natriuretic hormones iscardiodilatin, also referred to in literature as atrial natriureticpeptide (CDD/ANP). The isolation of cardiodilatin and the preparation ofbiologically active fragments of cardiodilatin are known from U.S. Pat.No. 4,751,284 (cf., W. G. Forssmann et al., Klin. Wochenschr. 1986, 64(Suppl. VI), 4-12). A review on isolation and characterization ofcardiodilatin and fragments thereof, as well as their physiologicalproperties has been published in Eur. J. Clin. Invest. 1986, 16; 439-451(W. G. Forssmann). From EP 0,349,545, a specific cardiodilatin fragmenthaving a chain length of 32 amino acids is known. Meanwhile, thisfragment is also referred to in literature as urodilatin (INN:ularitide). Furthermore, U.S. Pat. No. 5,354,900 (Suntory) describes abiologically active fragment having a chain length of 28 amino acids,known as α-hANP. Further biologically active cardiodilatin fragments orderivatives thereof have been described in EP 0,180,615. Therein, inparticular, cardiodilatin fragments are described which begin with theamino acid position Arg¹⁰² at the N-terminus and end with the amino acidposition Arg¹²⁵ or Arg¹²⁶ at the C-terminus. Instead of the designationcardiodilatin, the literature frequently uses the designation “atrialnatriuretic peptide” (ANP). In the numbering of the sequences of thecardiodilatin amino acids used in the following, reference is made tothe nomenclature used for the ANF/CDD (1-126) peptide (=ANP) in EP0,349,545.

A common structural feature of all hitherto known biologically activecardiodilatin fragments is the formation of a disulfide bridge betweenthe amino acids Cys¹⁰⁵ and Cys¹²¹, resulting in a stable ring of 17amino acids. It is believed that the formation of this ring issubstantially responsible for the biological activity of thecardiodilatin derivatives. At position Cys¹⁰⁵, the cardiodilatinfragments are substituted by an amino acid chain R¹ having a chainlength of 0-15 amino acids, and at position Cys¹²¹ by a chain R² havinga chain length of 0-5 amino acids. In the [SEQ ID NO. 1], the centralregion ANP(105-121) is presented in linearized form.

The cardiodilatin fragment ANP(95-126), with the INN designationularitide, is a particularly stable and biologically active humanpeptide, having diuretic activity and a relaxing effect on the smoothvascular muscles, which is formed of 32 amino acids and has thefollowing sequence, wherein both the cysteine amino acids at positions11 and 27 in the peptide are forming a disulfide bridge:

Urodilatin is found in human urine. EP 0,349,545 describes a process forrecovering urodilatin from urine using alginic acid, wherein thepeptides adsorbed to alginic acid are eluted, the eluate is fractionatedaccording to conventional purification methods, and the active fractionis recovered using a test based on the examination of the relaxingeffect of urodilatin on the smooth muscles.

Furthermore, EP 0,349,545 describes a stepwise chemical synthesis ofurodilatin using the Merrifield process (J. Am. Chem. Soc. 1963, 85;2149-2156), at a solid phase according to the ABI standard programfollowing the Boc strategy. In addition, this patent specificationdescribes the preparation of urodilatin from the partial fragmentANP(99-126). This fragment is bound to a solid phase, and is reactedwith a second partial fragment, the tetrapeptideBoc-Thr(But)-Ala-Pro-Arg(Tos). The peptide ANP(95-126) obtained from thecondensation is removed from the support, subjected to cyclization afterremoval of the protecting groups and subsequently, is processed andpurified in a per se known manner.

Similarly, EP 0,180,615 describes the chemical synthesis using a solidsupport, wherein formation of the cardiodilatin fragments describedtherein is effected successively, starting from the C-terminus indirection of the N-terminus. Here, condensation via partial fragments isnot described.

However, the cardiodilatin fragments prepared according to theprocedures described in literature did not have the purity necessary forclinical studies and for the authorization as medicinal product because,due to the synthesis, peptide impurities had been introduced into thefinal product which could not be removed even by subsequent purificationprocesses. Due to their immunogenic properties, the impurities may giverise to undesirable side-effects when administered to the patient, sothat therapeutic application involved risk. Moreover, the synthesiscould be accomplished at only a small scale under reasonable technicalinput and was not economically suitable for a larger production scale.Furthermore, another drawback of known processes for synthesis was theexisting potential risk of racemization due to which the urodilatin wasobtained with lower purity, lower biological activity and ininsufficient yield. Racemization of the product which frequently occurswith existing syntheses often resulted in insufficient optical purity ofthe final product, and these impurities frequently cannot be removed oronly with exceedingly high technical input.

Thus, it is an object of the invention to develop an improved processfor the chemical synthesis of cardiodilatin fragments which does notinvolve the above-mentioned drawbacks.

The object of the invention is attained by performing the synthesis ofcardiodilatin fragments on the basis of the Merrifield process using aspecific selection of peptide fragments.

Surprisingly, the course of synthesis has been found to be optimal whenthe cardiodilatin fragments are formed using three partial fragments,with the condensation of the partial fragments to give the cardiodilatinfragment of formula I being performed in such fashion that the formationis effected via condensation of partial fragments and bond formationbetween the amino acid positions Gly¹⁰⁸ and Arg¹⁰⁹ and the amino acidpositions Gly¹²⁰ and Cys¹²¹. This process is advantageous in that thecardiodilatin fragments of formula I can be obtained in higher yieldsand in higher purity as compared to the synthetic processes known fromprior art.

The synthesis of the cardiodilatin fragments of formula I is effected insuch way that initially, the three partial fragments having thesequences R¹-ANP(105-108), ANP(109-120) and ANP(121)-R² are preparedaccording to the Merrifield process. Then, preferably, condensation ofthe three partial fragments to give the cardiodilatin fragment offormula I is effected in two partial steps, whereby in a first step,condensation between the amino acid positions Gly¹²⁰ and Cys¹²¹ of thepartial fragments ANP(109-120) and Cys¹²¹-R² is effected, with theintermediate fragment ANP(109-121)-R² being formed. Then, in asubsequent second step, condensation of the thus obtained fragmentANP(109-121)-R² with the third partial fragment R¹-ANP(105-108) iseffected, forming the desired cardiodilatin fragment of formula I. Usingthe process according to the invention, the yield of cardiodilatinfragments is between 15 and 20%, based on the amount of eachcardiodilatin partial fragment used as starting material.

The three partial fragments having the sequences R¹-ANP(105-108),ANP(109-120) and ANP(121)-R² are prepared according to the Merrifieldprocess, wherein the amino acids with functional groups (hydroxy,carboxy, amino, or mercapto groups) present in the sequence aresubstituted by appropriate protecting groups. For example, as suitableprotecting groups the following groups are possible:

protecting groups for hydroxy groups: Boc (t-butyloxycarbonyl), tBu(t-butyl ether);

protecting groups for amino functions: Fmoc(9-fluorenylmethoxycarbonyl), Pbf(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl), Pmc(2,2,5,7,8-pentamethylchroman-6-sulfonyl), Trt (trityl);

protecting groups for carboxy groups: OtBu (t-butyl ester);

protecting groups for mercapto groups: Acm (acetamidomethyl) or Trt.

Here, the following protecting groups are preferred for the followingamino acids: tBu for the amino acids Thr, Asn, Tyr or Ser; Pbf or Pmcfor the amino acid Arg; Acm for the amino acid Cys; OtBu for the aminoacid Asp; Trt for the amino acids Gln, Asn or Cys.

Using the Fmoc strategy (B. Riniker et al., Tetrahedron 1993, 49;9307-9320), the protected partial fragments ANP(109-120),R¹-ANP(105-108) and ANP(121)-R² are formed on a solid support material.All the materials generally used in the Merrifield synthesis may serveas solid support materials. Preferred as support material is polystyrenefunctionalized as aminomethyl or benzhydrylamino compound. Thesuperacid-sensitive bonding of the peptide fragments to the resin bymeans of the 4-(4-hydroxymethyl-3-methoxyphenoxy)butyric acid linkerallows their removal without impeding the side-chain protection. Thefragments are purified by digestion with various solvents. Thus, thethree starting fragments ANP(109-120), R¹-ANP(105-108) and ANP(121)-R²are obtained with a C-terminal free carboxyl group and in good purity.When forming the peptides on the support resin, the yield in everysingle step of addition of one amino acid is nearly quantitative and isabout 97-99%

The flow diagram in FIG. 1 illustrates the principle of synthesis, withurodilatin ANP(95-126) as an example. Here, condensation of the fragmentBoc-1-14-OH (1) [this nomenclature corresponds to the generaldesignation of fragment R¹-ANP(105-108), wherein R¹=ANP(95-104)] withthe fragment H-15-32-OtBu (5) [corresponding to an ANP nomenclature ofANP(109-121)-R², wherein R²=ANP(122-126)] is effected. This fragment (5)is synthesized from the fragments Fmoc-15-26-OH (2) [corresponding to anANP nomenclature of ANP(109-120)] and H-27-32-OtBu (3c) [correspondingto an ANP nomenclature of ANP(121)-R²]. FIG. 2 represents the fragmentssynthesized and modified with protecting groups.

In the next step, the carboxyl group of fragment (3a) is converted tothe t-butyl ester (3b) (cf., Riniker et al., 22nd Europ. PeptideSymposium Interlaken, September 1992 (L7)). Subsequent removal of theFmoc group from fragment (3b) leads to the product (3c). This is fusedwith fragment (2), resulting in fragment (4). Removal of the Fmocprotecting group and condensation of the obtained fragment (5) withfragment (1) leads to the fully protected urodilatin (6). Removal of theprotecting groups by treatment with trifluoroacetic acid and1,3-propanedithiol as a scavenger provides the linear peptide (7) whichis cyclized to crude urodilatin (8) by oxidation with iodine solution.This is desalted, purified and may be lyophilized subsequently. Thesynthesis of other cardiodilatin fragments is conducted in an analogousfashion.

The synthesis according to the invention, involving the describedpartial fragments ANP(109-120), R¹-ANP(105-108) and ANP(121)-R² may beapplied to all the cardiodilatin fragments of formula I. In particular,cardiodilatin fragments are possible, wherein R¹ has a chain length of0-15 amino acids of the sequence ANP(90-104) or fragments thereof.Preferred for R¹ are chain lengths of 1-15 or 3-10 amino acids,particularly the sequences ANP(95-104), ANP(99-104) and ANP(102-104). Inparticular, the group R² represents a chain length of 1-5 amino acids ofthe sequence ANP(122-126) or fragments thereof. Preferably, however, thesequences ANP(122-126) and ANP(122-125) are possible for R².

Preferably, the cardiodilatin fragments ANP(95-126), ANP(99-126) andANP(102-126) may be prepared according to the process of the invention.The cardiodilatin fragments prepared by means of the process of theinvention, as well as the partial fragments required for condensationhave high optical purity in the range of about 96-99.9%, particularlyabout 98-99%.

Similarly, the synthesis is suitable for all the other derivatives ofcardiodilatin fragments wherein one or more amino acids in the sequenceof human ANP are replaced by other amino acids. In this meaning,replacement of amino acids includes corresponding substitutions,deletions or insertions of amino acids. For example, single or multipleamino acids may be replaced by the corresponding D-amino acids (cf., EP0,180,615). Likewise, peptides of similar structure and with acorresponding cyclic basic structure of 15-20 amino acids may beprepared in this way. Examples of such peptides are BNP (brainnatriuretic peptide) or CNP (C-type natriuretic peptide). The structuresof these peptides are described in J. Hypertension 1994, 12; 329-336 (N.C. Davidson and A. D. Struthers).

Likewise, the present invention is directed to novel partial fragmentsof ANP which are utilized for the preparation of cardiodilatin fragmentsof formula I according to the process of the invention.

More specifically, corresponding peptide fragments are those of the typeR¹-ANP(105-108), wherein R¹ represents an amino acid chain of sequenceANP(90-104) or fragments thereof having a chain length of 0-15 aminoacids, as well as their derivatives modified by protecting groups. Here,in particular, R¹ has the above-mentioned meanings. Another novelpeptide fragment is the fragment having the amino acid sequenceANP(109-120), as well as its derivatives modified by protecting groups,which is employed as a starting material in the condensation with thepartial fragment ANP(121)-R². Likewise, the corresponding ANP(121)-R²type peptide fragments represent a novelty and a subject matter of theinvention, wherein R² represents an amino acid chain of sequenceANP(122-126) or fragments thereof having a chain length of 0-5 aminoacids, as well as their derivatives modified by protecting groups. Inparticular, R² has the previously mentioned meaning. In addition, theinvention is directed to the intermediate ANP(109-121)-R² which isformed from the condensation reaction of the partial fragmentsANP(109-120) and ANP(121)-R² effected in the first reaction step.

Furthermore, the present invention relates to a process for preparinghigh-purity cardiodilatin fragments of formula I. Conventional syntheticprocesses and subsequent purification procedures on cardiodilatinfragments suffered from the drawback that in many cases a peptide purityin a range of merely 97-98% could be achieved.

EP 0,349,545 describes a purity level of about 98% in the case ofurodilatin; therein, the amount of urodilatin prepared was merely on asmaller laboratory scale in the range of a few milligrams. Thepurification procedure described in Example 5 therein is based on achromatography on a LH column (eluant: 1% AcOH, 1% TFEtOH) andsubsequent chromatography on a TSK column (Fractogel TSK-HW 40), whereinan aqueous solution of 10% AcOH and 1% TFETOH was used as the eluant. Ina final purification step, purification using preparative HPLC iseffected, without any further indications on the eluant being made.Within the scope of later experiments on the preparation of largeramounts of urodilatin in the range of a few grams for performingclinical tests, it was determined, however, that in spite of multiplepurification steps, the synthesized material could not be purifiedbeyond a purity level of more than 98%.

A comparable situation resulted in the case of cardiodilatin fragmentsdescribed in EP 0,180,615. Therein, for example, the purification forfragment ANP(102-126) in Example III.A.3 referred to ashANVP(127-151)—by chromatography on a type G25F Sephadex column isdescribed, where 0.5 M AcOH was used as the eluant. In a subsequentpurification step by means of ion exchange chromatography on CMSepharose or CM Cellulose using a solvent gradient of 0.01 M NH₄OAc/300mM NH₄OAc at pH 4.5, the peptide is obtained in a purity of about 97%.Likewise, this purity achieved is not satisfactory for the requirementsin drug manufacturing.

Surprisingly, it has been found that high-purity cardiodilatin fragmentsof formula I can be prepared if the crude product is purified using areversed-phase HPLC column, and the cardiodilatin fragment is elutedusing a buffer system containing triethylammonium phosphate (TEAP) andacetonitrile in aqueous solution. Here, preferably, the pH value of theelution buffer is adjusted to a value of 2-5, more specifically, of 2-3.Preferably, a type C₁₈ column, for example, Biotage module type filledwith YMC C₁₈ is used as the reversed-phase HPLC column. This column isequilibrated with triethylammonium phosphate buffer prior to loading thecardiodilatin fragments to be purified. For example, a solution of10-200 mM TEAP, preferably 50 mM TEAP, is employed as a suitable buffersolution. The amount of buffer for column equilibration depends on thecolumn size and this, in turn, on the amount of peptide to be purified.According to experience, a column volume of 75×300 mm (diameter×length)is required to purify an amount of peptide of 3-8 g of crude peptide. Inthis case, about 300 ml of a 50 mM TEAP buffer solution is required forequilibration. Subsequently, a solution of the concentrated crudeproduct of cardiodilatin fragment is applied. As a solvent, for example,10% acetic acid is suitable. Thereafter, the peptide is eluted in acontinuous gradient by continuous charging of eluant (mixture of anaqueous solution of 10-200 mM TEAP and acetonitrile at a volume ratio of2:3; pH 2-5). Elution of peptide is particularly advantageous if acontinuous gradient of eluant is applied, where 22-28% of solventgradient is used for a period of 90 minutes, followed by 28% of solventgradient for 10 minutes and, eventually, 28-40% of gradient for 20minutes. Preferably, the flow rate is 100-200 ml/min, more specifically,about 140 ml/min. In the meaning of the purification process accordingto the invention, a buffer mixture of triethylammonium phosphate inwater and acetonitrile at a mixing ratio of from 1:3 to 2:1 (v/v), morespecifically of about 2:3 (v/v) is used as elution buffer. The pH valueof the buffer solution is 2-5, preferably 2-3, and more specificallyabout 2.25. TEAP may be used at a concentration of 10-200 mM, preferably20-100 mM, and more specifically, of about 50 mM. According to theinvention, optimum separation is achieved in the reversed-phase HPLC byequilibrating the column using 50 mM TEAP, pH 2.25, and eluting thepeptide with a buffer consisting of 50 mM TEAP, pH 2.25, andacetonitrile at a ratio of 2:3.

Conventional purification procedures using aqueous 0.1% trifluoroaceticacid (TFA), for example, are not capable of further separating the polarimpurities contained in the crude products, as are revealed in FIG. 5 inthe example of urodilatin (FIG. 6). In contrast, in the case of theeluants used according to the invention, there is significant separationof both impurities (see FIG. 5). Furthermore, use of the eluantaccording to the invention is advantageous in that the base line in theHPLC chromatogram takes an absolutely steady course, while in the caseof TFA, a strong drift can be observed. In addition, use of TFA suffersfrom the drawback that a higher back pressure builds up on the HPLCcolumn, which is not the case for the eluant according to the invention.

Using the process according to the invention, high-purity cardiodilatinfragments of formula I are obtained in a purity of at least 99% andpreferably, of up to 99.9%. Optionally, the cardiodilatin fragments maysubsequently be converted to their physiologically acceptable salts,such as the acetate or citrate salts. The cardiodilatin fragmentsobtained are substantially free of peptide impurities so that not onlythe reversed-phase HPLC exhibits a single peak but also the much moresensitive method of capillary electrophoresis (CE) provides a singlemigration peak. In the case of urodilatin, the latter shows a mass of3505.9±1 in the MS analysis, without byproducts being detected. Itturned out that the use of capillary electrophoresis allows an excellentdemonstration of the differences between cardiodilatin fragmentsobtained according to prior art and the cardiodilatin fragmentsaccording to the invention. FIG. 3 illustrates the CE chromatogram of aurodilatin production batch produced according to prior art. Herein, itcan be clearly seen that the product still contains impurities. Incontrast, FIG. 4 represents the CE chromatogram of a urodilatinproduction batch produced according to the process of the invention andpurified correspondingly. It is clearly obvious that the product issubstantially free of other peptide impurities and exhibits a singlemigration peak in the capillary electrophoresis.

Therefore, the invention is directed to high-purity cardiodilatinfragments of formula I which are remarkable in that they do not containsubstantial peptide impurities detectable by capillary electrophoresisand MS analysis, and that the purity analysis using capillaryelectrophoresis exhibits a single migration peak.

Similarly, the purification procedure according to the invention is alsosuitable for the preparation of analogous high-purity peptide compoundssuch as, e.g., BNP (brain natriuretic peptide), CNP(C-type natriureticpeptide) or derivatives thereof. The cyclic structure of ANP is based onthe oxidation of two cysteine residues within the amino acid sequence,forming a cyclic ring of 17 amino acids. Other peptides which also formthe characteristic cyclic structure of 15-20 amino acids, particularly17 amino acids, such as, e.g., BNP or CNP, may be converted to thehigh-purity forms in the same fashion using the purification procedureaccording to the invention.

In the following embodiments, the invention will be illustrated usingthe selected representative cardiodilatin fragments ANP(95-126),ANP(99-126) and ANP(102-126).

EXAMPLE 1

General Procedures of Solid-Phase Synthesis According to the MerrifieldProcess

a) Solid-Phase Synthesis on a Support Resin

Starting from the C-terminus of the peptide to be synthesized, the firstamino acid (AA) protected by the Fmoc group at the N-terminal end, isbound to the support resin (Fmoc-AA-OHMPB-support resin). With astandard batch of 6.66 mmoles, the Fmoc protecting group is subsequentlyremoved by adding 100 ml of a solvent mixture of piperidine andN-methylpyrrolidine (1:4 v/v). Then, the resin suspension is stirred for10 minutes, subsequently filtrated, and again, 100 ml of the piperidineand NMP solvent mixture is added. Then, the suspension is stirred for 10minutes, filtrated and subsequently washed with NMP an isopropanol, andcompleteness of the reaction is checked using the Kaiser test.

Thereafter, the next amino acid is coupled to the resin. Initially, 20mmoles of a 0.5 M solution of diisopropylethanylamine (DIPEA) in NMP isadded to the resin, then 2.5 mmoles of a 0.5 M solution of1-hydroxybenzotriazole (OHBT) in NMP, followed by 10 mmoles of the aminoacid to be coupled in 25 ml of NMP. Thereafter, 11 mmoles of a 0.25 Msolution of TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate) in NMP is added and stirred for 10 minutes.Completeness of the reaction is checked using the Kaiser test.Subsequently, the resin is filtrated and washed with NMP.

This process is continued in the same way, until the peptide chain ofdesired chain length of amino acids is built up on the resin. Whensynthesis is complete, the resin is dried to constant weight at 40° C.

b) Removal of the Protected Peptides from the Support Resin

Each of 10 suction flasks is charged with 75 ml of methanol and 3 ml ofpyridine. 50 g of the support resin prepared according to step a) isstirred 10 times with 250 ml of 1% TFA in dry methylene chloride for oneminute on the suction funnel, and is filtrated directly into therespective suction flask. These 10 filtrates are checked using thinlayer chromatography. Fractions containing product are combined andevaporated to dryness. The residue is triturated with deionized water,and the crystalline residue is filtrated off and dried.

EXAMPLE 2

Preparation of Fragment ANP(109-120)

Following the general procedures of Example 1, and starting from 273 gof Fmoc-Gly-OHMPB-support resin (corresponding to 130 mmoles), 170.3 gof the fully protected cardiodilatin fragment ANP(109-120) is obtained.

EXAMPLE 3

Preparation of Fragment ANP(121-126)

Following the general procedures of Example 1, and starting from 264 gof Fmoc-Tyr-OHMPB-support resin (corresponding to 115 mmoles), 150.7 gof the fully protected cardiodilatin fragment ANP(121-126) is obtained.Here, the N-terminal end of the fragment is protected by the Fmoc group.

Subsequently, the terminal hydroxy group at the C-terminal end of thefragment is converted to the OtBu protecting group. For esterification,149 g of the fully protected fragment is dissolved in 500 ml oftrifluoroethanol and 4.1 l of chloroform. This is followed by additionof 141 ml of TBTA (t-butyl-2,2,2-trichloroacetimidate), and the solutionis heated at reflux for one hour. After the reaction is completed, thesolution is concentrated to give a crystalline-oily residue, 6.8 l ofdiisopropyl ether is added, and the suspension is stirred at roomtemperature for 14 hours. The product is filtrated off and dried toconstant weight. 136.7 g of fragment 3b indicated in FIG. 2 is obtained.

Subsequently, the Fmoc protecting group at the N-terminal end of thefragment is removed, and conversion to fragment 3c indicated in FIG. 2is effected. To this end, a solution of fragment 3b (135.7 g) in 1.8 lof DMF and 74 ml of diethylamine is stirred at room temperature for 3hours. The solution is evaporated to complete dryness in a vacuum. Theresidue is digested with 1.4 l of deionized water and filtrated off. Thewet product is taken up in 3 l of MTBE (methyl t-butyl ether). Thesolution is extracted with a saturated NaCl solution (2×100 ml), and theorganic phase is dried with sodium sulfate. The solution is concentratedto a volume of 500 ml. Following addition of 1.5 l of isopropyl ether,stirring for two hours is effected. The product is filtrated and dried.The yield is 104.6 g of fragment 3c indicated in FIG. 2.

EXAMPLE 4

Preparation of Fragment ANP(121-125)

In an analogous manner as described in Example 3, starting from 264 g ofFmoc-Arg(Pbf)-OHMPB-support resin and following the procedure described,115.1 g of cardiodilatin fragment ANP(121-125) is obtained.

EXAMPLE 5

Preparation of Fragment ANP(95-108)

Following the general procedures of Example 1, and starting from 210 gof Fmoc-Gly-OHMPB-support resin, 151.5 g of the fully protectedcardiodilatin fragment ANP(95-108) is obtained.

EXAMPLE 6

Preparation of Fragment ANP(99-108)

Following the general procedures of Example 1, and starting from 190 gof Fmoc-Gly-OHMPB-support resin, 145.1 g of the fully protectedcardiodilatin fragment ANP(99-108) is obtained.

EXAMPLE 7

Preparation of Fragment ANP(102-108)

Following the general procedures of Example 1, and starting from 220 gof Fmoc-Gly-OHMPB-support resin, 165.3 g of the fully protectedcardiodilatin fragment ANP(102-108) is obtained.

EXAMPLE 8

Condensation of the Partial Fragments to the Intermediate Product

The fragment ANP(109-120) is converted to the intermediateANP(109-121)-R² by condensation with the C-terminal fragment ANP(121)-R²according to the following general process:

The fragment ANP(109-120), the amino terminus of which is protected bythe Fmoc group, is dissolved in N-methylpyrrolidone. Subsequently, TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),1-hydroxybenzotriazole and diisopropylethylamine are added to thesolution at room temperature with stirring. Thereafter, the fragmentANP(121)-R² provided with an appropriate protecting group at theC-terminal end and dissolved in N-methylpyrrolidone is added to thesolution. In the following, the reaction is monitored by thin layerchromatography. After about 2 hours, the reaction is complete. Then, thereaction mixture is dripped onto diisopropyl ether with stirring andsubsequently stirred for about 30 minutes. The precipitate is filtratedon a porcelain suction funnel over hard filter and washed twice withdiisopropyl ether. Thereafter, the residue is suspended in acetonitrileand digested at room temperature with stirring. Subsequently, theproduct is filtrated on a porcelain suction funnel, rewashed withacetonitrile and dried to constant weight in a vacuum chamber at 40° C.The thus obtained crude product represents the cardiodilatin fragmentFmoc-ANP(109-121)-R² protected at the amino terminus by the Fmocprotecting group. Thereafter, the Fmoc group is removed according toknown procedures to obtain the intermediate product H-ANP(109-121)-R².

EXAMPLE 9

Condensation of Fragments ANP(109-120) with ANP(121-126) to ANP(109-126)

Following the general procedure described in Example 8, 21.6 g ofFmoc-ANP(109-120) is dissolved in 650 ml of N-methylpyrrolidone.Subsequently, 3.2 g of TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),1.5 g of 1-hydroxybenzotriazole and 3.5 ml of diisopropylethylamine areadded to the solution at room temperature with stirring. Thereafter, asolution of H-ANP(121-126)-OtBu, dissolved in 150 ml ofN-methylpyrrolidone, is added. In the following, the reaction ismonitored by thin layer chromatography. After about 2 hours, thereaction is complete. Then, the reaction mixture is dripped onto 4 l ofdiisopropyl ether with stirring and subsequently stirred for about 30minutes. The precipitate is filtrated on a porcelain suction funnel overhard filter and washed twice with 500 ml of diisopropyl ether.Thereafter, the residue is suspended in 600 ml of acetonitrile anddigested at room temperature with stirring. Subsequently, the product isfiltrated on a porcelain suction funnel, rewashed with 300 ml ofacetonitrile and dried to constant weight in a vacuum chamber at 40° C.Subsequently, the crude product Fmoc-ANP(109-126) thus obtained in anamount of 32.3 g is converted to the unprotected ANP(109-126) byaddition of diethylamine. The yield is 30.2 g.

EXAMPLE 10

Condensation of Fragments ANP(109-120) with ANP(121-125) to ANP(109-125)

Following the general procedure described in Example 8, 18.6 g ofFmoc-ANP(109-120) is dissolved in 600 ml of N-methylpyrrolidone.Subsequently, 3.0 g of TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),1.2 g of 1-hydroxybenzotriazole and 3.0 ml of diisopropylethylamine areadded to the solution at room temperature with stirring. Thereafter, asolution of H-ANP(121-125)-OtBu, dissolved in 150 ml ofN-methylpyrrolidone, is added. In the following, the reaction ismonitored by thin layer chromatography. After about 2 hours, thereaction is complete. Then, the reaction mixture is dripped onto 4 l ofdiisopropyl ether with stirring and subsequently stirred for about 30minutes. The precipitate is filtrated on a porcelain suction funnel overhard filter and washed twice with 450 ml of diisopropyl ether.Thereafter, the residue is suspended in 500 ml of acetonitrile anddigested at room temperature with stirring. Subsequently, the product isfiltrated off on a porcelain suction funnel, rewashed with 250 ml ofacetonitrile and dried to constant weight in a vacuum chamber at 40° C.Subsequently, the crude product Fmoc-ANP(109-125) thus obtained in anamount of 29.1 g is converted to the unprotected ANP(109-125) byaddition of diethylamine. The yield is 28.2 g.

EXAMPLE 11

Condensation of the Partial Fragments to the Final Product

The intermediate ANP(109-121)-R² is converted to the final productR¹-ANP(105-121)-R² by condensation with the amino-terminal fragmentR¹-ANP(105-108) according to the following general process:

The fragment R¹-ANP(105-108), the amino terminus of which is protectedby an appropriate protecting group, is dissolved in N-methylpyrrolidone.Subsequently, TBTU (2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroborate), 1-hydroxybenzotriazole and diisopropylethylamine areadded to the solution at room temperature with stirring. Thereafter, thefragment ANP(109-121)-R² provided with an appropriate protecting groupat the C-terminal end and dissolved in N-methylpyrrolidone is added tothe solution. In the following, the reaction is monitored by thin layerchromatography. After about 2 hours, the reaction is complete. Then, thereaction mixture is dripped onto diisopropyl ether with stirring andsubsequently stirred for about 30 minutes. The precipitate is filtratedon a porcelain suction funnel over hard filter and washed twice withdiisopropyl ether. Thereafter, the residue is suspended in acetonitrileand digested at room temperature with stirring. Subsequently, theproduct is filtrated off on a porcelain suction funnel, rewashed withacetonitrile and dried to constant weight in a vacuum chamber at 40° C.The thus obtained crude product represents the cardiodilatin fragmentR¹-ANP(105-121)-R² protected by appropriate protecting groups at theamino terminus and the C-terminus. Thereafter, the protecting group isremoved according to known procedures to obtain the intermediate productH—R¹-ANP(109-121)-R². Following complete removal of the protectinggroups, the obtained cardiodilatin fragment is converted to the cyclizedderivative by oxidation and according to known procedures, for example,using iodine.

EXAMPLE 12

Condensation of Fragments ANP(109-126) and ANP(95-108) to ANP(95-126)

a) Preparation of ANP(95-126)

Following the general procedure described in Example 11, 20.6 g ofBoc-ANP(95-108) is dissolved in 400 ml of N-methylpyrrolidone.Subsequently, 2.7 g of TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),1.3 g of 1-hydroxybenzotriazole and 2.7 ml of diisopropylethylamine areadded to the solution at room temperature with stirring. Thereafter, asolution of 29.4 g of H-ANP(109-126)-OtBu, dissolved in 400 ml ofN-methylpyrrolidone, is added. In the following, the reaction ismonitored by thin layer chromatography. After about 2 hours, thereaction is complete. Then, the reaction mixture is dripped onto 6.5 lof diisopropyl ether with stirring and subsequently stirred for about 30minutes. The precipitate is filtrated on a porcelain suction funnel overhard filter and washed twice with 500 ml of diisopropyl ether.Thereafter, the residue is suspended in 600 ml of acetonitrile anddigested at room temperature with stirring. Subsequently, the product isfiltrated off on a porcelain suction funnel, rewashed with 500 ml ofacetonitrile and dried to constant weight in a vacuum chamber at 40° C.Subsequently, the crude product Boc-ANP(95-126)-OtBu thus obtained in anamount of 42.5 g is converted to the unprotected ANP(95-126) and dried.The yield is 27.5 g.

b) Cyclization of the Deprotected Linear ANP(95-126)

60 g of unprotected ANP(95-126) is dissolved in 16 l of 5% acetic acidin deionized water (v/v) and oxidized by addition of 570 ml of a 0.02 Mmethanolic iodine solution. The reaction is complete after 5 minutes.Excess iodine is destroyed by addition of a 0.1 M sodium thiosulfatesolution. The cyclization solution obtained is subjected directly tofurther processing.

EXAMPLE 13

Condensation of Fragments ANP(109-126) and ANP(99-108) to ANP(99-126)

Analogous to the procedure described in Example 12, 22.5 g ofBoc-ANP(99-108) is dissolved in 400 ml of N-methylpyrrolidone.Subsequently, 2.9 g of TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),1.4 g of 1-hydroxybenzotriazole and 2.8 ml of diisopropylethylamine areadded to the solution at room temperature with stirring. Thereafter, asolution of 30.6 g of H-ANP(109-126)-OtBu, dissolved in 400 ml ofN-methylpyrrolidone, is added. In the following, the reaction ismonitored by thin layer chromatography. After about 2 hours, thereaction is complete. Then, the reaction mixture is dripped onto 6.5 lof diisopropyl ether with stirring and subsequently stirred for about 30minutes. The precipitate is filtrated on a porcelain suction funnel overhard filter and washed twice with 500 ml of diisopropyl ether.Thereafter, the residue is suspended in 600 ml of acetonitrile anddigested at room temperature with stirring. Subsequently, the product isfiltrated off on a porcelain suction funnel, rewashed with 500 ml ofacetonitrile and dried to constant weight in a vacuum chamber at 40° C.Subsequently, the crude product Boc-ANP(99-126)-OtBu thus obtained in anamount of 44.7 g is converted to the unprotected ANP(99-126) and dried.The yield is 28.1 g.

EXAMPLE 14

Condensation of Fragments ANP(109-126) and ANP(102-108) to ANP(102-126)

Analogous to the procedure described in Example 12, 20.4 g ofBoc-ANP(102-108) is dissolved in 360 ml of N-methylpyrrolidone.Subsequently, 2.7 g of TBTU(2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate),1.4 g of 1-hydroxybenzotriazole and 2.6 ml of diisopropylethylamine areadded to the solution at room temperature with stirring. Thereafter, asolution of 30.1 g of H-ANP(109-126)-OtBu, dissolved in 400 ml ofN-methylpyrrolidone, is added. In the following, the reaction ismonitored by thin layer chromatography. After about 2 hours, thereaction is complete. Then, the reaction mixture is dripped onto 6.5 lof diisopropyl ether with stirring and subsequently stirred for about 30minutes. The precipitate is filtrated on a porcelain suction funnel overhard filter and washed twice with 500 ml of diisopropyl ether.Thereafter, the residue is suspended in 600 ml of acetonitrile anddigested at room temperature with stirring. Subsequently, the product isfiltrated off on a porcelain suction funnel, rewashed with 500 ml ofacetonitrile and dried to constant weight in a vacuum chamber at 40° C.Subsequently, the crude product Boc-ANP(102-126)-OtBu thus obtained inan amount of 41.2 g is converted to the unprotected ANP(102-126) anddried. The yield is 26.9 g.

EXAMPLE 15

Purification of ANP(95-126) and Preparation of the High-Purity Form

a) Concentrating the Cyclized Urodilatin [ANP(95-126)]

The cyclization solution (about 17 liters of 5% AcOH, in deionized water(v/v), contains about 60 g of cyclized urodilatin) is applied (flow rate130 ml/min) on a glass column (diameter: 70 mm, length: 900 mm, filledwith Vydac 218 TPB 2030) equilibrated with 1000 ml of buffer A3 (0.1%TFA (v/v) in deionized water).

Once application by pumping is finished, the peptide is eluted bycontinuous charging of buffer B3 (0.1% TFA in deionized water/ACN 2:3v/v) in a continuous gradient (0% buffer B during 40 min; 15-35% bufferB during 90 min; 35% buffer B during 10 min; flow rate 130 ml/min).

urodilatin fractions showing a purity of more than 75% on monitoring byanalytical HPLC are combined. These combined fractions are diluted withone volume equivalent of deionized water and applied (flow rate 140ml/min) on a Biotage module (diameter: 75 mm, length: 300 mm, filledwith YMC C₁₈, 120 A, 10 μm) equilibrated with 300 ml of buffer A3.

Subsequently, the concentrated peptide is eluted by washing the columnwith 100% buffer B3, and the acetonitrile is evaporated. The remainingsolution is lyophilized.

Between 17 and 20 g of urodilatin with a purity of more than 90% isobtained.

b) Purification of the Concentrated Urodilatin

4.5 g of the concentrated urodilatin is dissolved in 250 ml of 10′, AcOHin deionized water (v/v) and applied (flow rate 140 ml/min) on a Biotagemodule (diameter: 75 mm, length: 300 mm, filled with YMC C₁₈, 120 A, 10μm) equilibrated with 300 ml of buffer A4 (50 mM TEAP, pH 2.25, indeionized water).

The peptide is eluted by continuous charging of buffer B4 (50 mM TEAP,pH 2.25 in deionized water/ACN 2:3 v/v) in a continuous gradient (22-28%B during 90 min; 28% B during 10 min; 28-40% B during 20 min; flow rate140 ml/min).

urodilatin fractions showing a purity of more than 99% and impurities ofnot more than 0.5% on monitoring by analytical HPLC are combined. Thesecombined fractions are diluted with one volume equivalent of deionizedwater and pumped onto the Biotage module previously cleaned with 1000 mlof buffer B3 and subsequently equilibrated with 300 ml of buffer A3. Fordesalting, a washing with 1200 ml of buffer A3 is made.

The pure product is eluted by washing the column with 1500 ml of bufferB3, and the acetonitrile is evaporated. The remaining solution islyophilized.

The result is between 2.3 and 2.7 g of high-purity urodilatin.

c) Resalting of Urodilatin×TFA to Urodilatin Acetate

2.5 g of high-purity urodilatin×TFA salt is dissolved in 80 ml of 5%AcOH, in deionized water v/v, and applied to a chromatography column(diameter: 20 mm, length: 300 mm, filled with 40 ml of Merck ionexchanger III acetate form) washed with 5% AcOH. A washing with 40 ml of5% AcOH is made. The eluate, about 125 ml, is applied once more to thesame ion exchange column. A washing with 55 ml of 5% AcOH is made. Theeluate, about 180 ml, is filtrated clear over a polysulfone membrane(diameter 47 mm, 0.2 μm). The solution is lyophilized.

The result is between 2.05 and 2.30 g of high-purity urodilatin acetate.

EXAMPLE 16

Purification of ANP(99-126) and Preparation of the High-Purity Form

a) Concentrating the Cyclized Cardiodilatin fragment ANP(99-126)

Analogous to Example 15a), the cyclization solution (about 15 liters of5% AcOH, in deionized water (v/v), with a peptide content of about 50 g)is applied (flow rate 130 ml/min) on a glass column equilibrated with1000 ml of buffer A3 (0.1% TFA (v/v) in deionized water). Onceapplication by pumping is finished, the peptide is eluted by continuouscharging of buffer B3 (0.1% TFA in deionized water/ACN 2:3 v/v) in acontinuous gradient (0% buffer B during 40 min; 15-35% buffer B during90 min; 35% buffer B during 10 min; flow rate 130 ml/min). Peptidefractions showing a purity of more than 75% on monitoring by analyticalHPLC are combined. These combined fractions are diluted with one volumeequivalent of deionized water and applied (flow rate 140 ml/min) on aBiotage module equilibrated with 300 ml of buffer A3. Subsequently, theconcentrated peptide is eluted by washing the column with 100% bufferB3, and the acetonitrile is evaporated. The remaining solution islyophilized.

The result is between 14 and 17 g of cardiodilatin fragment ANP(99-126)with a purity of more than 90%.

b) Purification of the Concentrated ANP(99-126)

3.5 g of the cardiodilatin fragment concentrated according to Example16a) is dissolved in 200 ml of 10% AcOH in deionized water (v/v) andapplied (flow rate 140 ml/min) on a Biotage module equilibrated with 300ml of buffer A4 (50 mM TEAP, pH 2.25, in deionized water). The peptideis eluted by continuous charging of buffer B4 (50 mM TEAP, pH 2.25 indeionized water/ACN 2:3 v/v) in a continuous gradient (22-28% B during90 min; 28% B during 10 min; 28-40% B during 20 min; flow rate 140ml/min).

Peptide fractions showing a purity of more than 99% and impurities ofnot more than 0.5% on monitoring by analytical HPLC are combined. Thesecombined fractions are diluted with one volume equivalent of deionizedwater and pumped onto the Biotage module previously cleaned with 1000 mlof buffer B3 and subsequently equilibrated with 300 ml of buffer A3. Fordesalting, a washing with 1000 ml of buffer A3 is made.

The pure product is eluted by washing the column with 1500 ml of bufferB3, and the acetonitrile is evaporated. The remaining solution islyophilized.

The result is between 1.7 and 2.2 g of high-purity cardiodilatinfragment ANP(99-126). Analogous to the procedure described in Example14c), this fragment is converted to the corresponding acetate salt. Theresult is between 1.3 and 1.7 g of high-purity ANP(99-126) acetate.

EXAMPLE 17

Purification of ANP(102-126) and Preparation of the High-Purity Form

a) Concentrating the Cyclized Cardiodilatin Fragment ANP(102-126)

Analogous to Example 15a), the cyclization solution (about 18 liters of5% AcOH, in deionized water (v/v), with a peptide content of about 65 g)is applied (flow rate 130 ml/min) on a glass column equilibrated with1000 ml of buffer A3 (0.1% TFA (v/v) in deionized water). Onceapplication by pumping is finished, the peptide is eluted by continuouscharging of buffer B3 (0.1% TFA in deionized water/ACN 2:3 v/v) in acontinuous gradient (0% buffer B during 40 min; 15-35% buffer B during90 min; 35% buffer B during 10 min; flow rate 130 ml/min). Peptidefractions showing a purity of more than 75% on monitoring by analyticalHPLC are combined. These combined fractions are diluted with one volumeequivalent of deionized water and applied (flow rate 140 ml/min) on aBiotage module equilibrated with 300 ml of buffer A3. Subsequently, theconcentrated peptide is eluted by washing the column with 100% bufferB3, and the acetonitrile is evaporated. The remaining solution islyophilized.

The result is between 19 and 23 g of cardiodilatin fragment ANP(102-126)with a purity of more than 90%.

b) Purification of the Concentrated ANP(102-126)

4.8 g of the cardiodilatin fragment concentrated according to Example17a) is dissolved in 200 ml of 10% AcOH in deionized water (v/v) andapplied (flow rate 140 ml/min) on a Biotage module equilibrated with 300ml of buffer A4 (50 mM TEAP, pH 2.25, in deionized water). The peptideis eluted by continuous charging of buffer B4 (50 mM TEAP, pH 2.25 indeionized water/ACN 2:3 v/v) in a continuous gradient (22-28% B during90 min; 28% B during 10 min; 28-40% B during 20 min; flow rate 140ml/min).

Peptide fractions showing a purity of more than 99% and impurities ofnot more than 0.5% on monitoring by analytical HPLC are combined. Thesecombined fractions are diluted with one volume equivalent of deionizedwater and pumped onto the Biotage module previously cleaned with 1000 mlof buffer B3 and subsequently equilibrated with 300 ml of buffer A3. Fordesalting, a washing with 1000 ml of buffer A3 is made.

The pure product is eluted by washing the column with 1500 ml of bufferB3, and the acetonitrile is evaporated. The remaining solution islyophilized.

The result is between 1.9 and 2.4 g of high-purity cardiodilatinfragment ANP(102-126). Analogous to the procedure described in Example14c), this fragment is converted to the corresponding acetate salt. Theresult is between 1.5 and 1.9 g of high-purity ANP(99-126) acetate.

EXAMPLE 18

Analytical HPLC Examinations Using the ANP(95-126) Example

a) Elution with TEAP Buffer, pH 2.25

50 μg of ANP(95-126) is injected onto an analytical HPLC column. Alinear gradient of buffer B of 25-45% during 20 minutes (buffer A: 50 mMTEAP, pH 2.25; buffer B: mixture of A and acetonitrile at a volume ratioof 2:3) served as the eluant. The chromatogram in FIG. 5 reveals thattwo polar impurities are contained which may be separated by the eluantemployed.

Legend to FIG. 5:

25-45% in 20 min.

Buffer A: 50 mM TEAP pH 2,25

Buffer B: A:ACN (2:3)

215 nm 1,0 ml/{acute over ( )}C—Nr. 4040465 C

M+N 250/1/4°/3 Nuc 300 A5 u C18

D-2500

Method: 50 μg; TAG 243 CH:1; Peak reject: 5000

File: 1; Calculation method: area %; Table: 0; conc: area No. RT Area %BC 5 7.82 53358 0.311 BV 6 8.08 84196 0.491 VV 7 9.07 386602 2.255 VV 89.78 1265799 7.384 VV 9 10.56 4701290 27.430 VV 10 10.92 10557085 61.582VV 11 11.91 27613 0.161 TBB 12 12.82 8763 0.051 TBB 13 13.76 14346 0.084BB 14 14.86 31959 0.186 BB 15 19.04 10892 0.064 BB Total 17143003 100.00b) Elution with 0.1% TFA (trifluoroacetic acid)

Analogous to Example 18a), 50 μg of ANP(95-126) same production batch isapplied onto an analytical HPLC column. A linear gradient of buffer B of30-50% during 20 minutes (buffer A: 0.1% TFA in water; buffer B: mixtureof A and acetonitrile at a volume ratio of 2:3) served as the eluant.The chromatogram in FIG. 6 reveals that separation of the containedimpurities by means of this eluant is not effected. Compared to thechromatogram in Example a), the main peak is broader and the isolatedproduct contains both of the polar impurities which can be recognized inthe chromatogram FIG. 5.

Legend to FIG. 6:

30-50% B in 20 min.

Buffer A: 0,1% TFA in water

Buffer B A:ACN (2:3)

215 nm 1,0 ml/{acute over ( )}C—Nr. 4011079 C

M+N 250/1/4°/3 Nuc 300 LA 5u C18

D-2500

Method: 50 μg; TAG 142; CH:1; Peak reject: 5000

File: 2; Calculation method: area %; Table: 0; conc: area No. RT Area %BC 2 3.64 5073 0.040 BV 4 5.10 6624 0.053 BB 5 5.92 8161 0.065 BB 6 7.366814 0.054 BB 7 9.11 252878 2.012 BB 9 11.73 87629 0.697 BB 10 12.60258273 2.055 BB 11 13.09 4578590 36.428 VV 12 13.26 7175177 57.086 VV 1314.67 179155 1.425 TBB 14 17.48 10611 0.084 BB Total 12568985 100.00

EXAMPLE 19

Purity Check by Capillary Electrophoresis

Lyophilized samples of the final products of cardiodilatin fragmentsfrom Examples 15 through 17 are dissolved in water at a concentration of1 mg/ml and analyzed immediately. Capillary electrophoresis wasperformed using the Beckman P/ACE 2100 system under the followingconditions:

Capillary: Fused Silica by Supelco, separation length 50 cm, internaldiameter 75 μm

Detection wave length: 200 nm

Injection period: 1 s

Separation buffer: 100 mM sodium phosphate, pH 2.5; 0.02%hydroxypropylmethylcellulose

Separation parameters: 25° C., 80 μA, 30 min

FIG. 3 shows the chromatogram obtained for prior art urodilatin.

FIG. 4 shows the chromatogram for high-purity urodilatin obtainedaccording to Example 15.

A comparison of both chromatograms reveals that the urodilatin accordingto the invention differs significantly from prior art urodilatin. Theurodilatin according to the invention is free of peptide impurities.

INDEX OF ABBREVIATIONS

Amino Acids

-   Ala L-Alanine-   Asn L-Asparagine-   Asp L-Asparaginic acid-   Arg L-Arginine-   Cys L-Cysteine-   Gin L-Glutamine-   Gly Glycine-   Ile L-Isoleucine-   Leu L-Leucine-   Met L-Methionine-   Phe L-Phenylalanine-   Pro L-Proline-   Ser L-Serine-   Thr L-Threonine-   Tyr L-Tyrosine    Protecting Groups-   Boc t-Butyloxycarbonyl-   Fmoc 9-Fluorenylmethoxycarbonyl-   OtBu t-Butyl ester-   Pbf 2,2,4,6,7-Pentamethyldihydrobenzofuran-5-sulfonyl-   Pmc 2,2,5,7,8-Pentamethylchroman-6-sulfonyl-   tBu t-Butyl ether-   Acm Acetamidomethyl-   Trt Trityl    Reagents/Solvents-   ACN Acetonitrile-   TFA Trifluoroacetic acid-   TEAP Triethylammonium phosphate

1-20. (canceled)
 21. A fragment of ANP (95-126) selected from the groupconsisting of: (a)Boc-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Acm)-Phe-Gly-Gly-OH(SEQ ID 6); (b)Fmoc-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-OH(SEQ ID 7); (c) Fmoc-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)OH(SEQ ID 8); (d)Fmoc-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-OtBu (SEQ ID 8);(e) H-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-OtBu (SEQ ID 8);(f)Fmoc-Arg(Pbf)-Met-Asp(Otbu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-OtBu(SEQ ID 9); and (g)H-Arg(Pbf)-Met-Asp(Otbu)-Arg(Pbf)-Ile-Gly-Ala-Gln(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-OtBu(SEQ ID 9).
 22. A fragment of ANP (95-126) selected from the groupconsisting of: (a)Boc-Thr(tBu)-Ala-Pro-Arg(Pbf)-Ser(tBu)-Leu-Arg(Pbf)-Arg(Pbf)-Ser(tBu)-Ser(tBu)-Cys(Acm)-Phe-Gly-Gly-Arg(Pbf)-Met-Asp(OtBu)-Arg(Pbf)-Ile-Gly-Ala-GIn(Trt)-Ser(tBu)-Gly-Leu-Gly-Cys(Trt)-Asn(Trt)-Ser(tBu)-Phe-Arg(Pbf)-Tyr(tBu)-OtBu-(SEQ ID 10); and (b)H-Thr-Ala-Pro-Arg-Ser-Leu-Arg-Arg-Ser-Ser-Cys(Acm)-Phe-Gly-Gly-Arg-Met-Asp-Arg-Ile-Gly-Ala-Gln-Ser-Gly-Leu-Gly-Cys-Asn-Ser-Phe-Arg-Tyr-OH(SEQ ID 5).
 23. The fragment of claim 22, wherein such fragment isstable.